RNAi-mediated inhibition of spleen tyrosine kinase-related inflammatory conditions

ABSTRACT

RNA interference is provided for inhibition of spleen tyrosine kinase (Syk) mRNA expression, in particular, for treating patients having a Syk-related inflammatory condition or at risk of developing a Syk-related inflammatory condition such as allergic conjunctivitis, ocular inflammation, dermatitis, rhinitis, asthma, allergy, or mast-cell disease.

The present application is a divisional of U.S. application Ser. No.12/296,621 filed Oct. 9, 2008 (now U.S. Pat. No. 8,048,863), which isthe National Stage of International Application Serial No.:PCT/US2007/066619 filed Apr. 13, 2007, which claims benefit to U.S.Provisional Patent Application Ser. No. 60/791,847 filed Apr. 13, 2006,the text of which is specifically incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of interfering RNAcompositions for silencing spleen tyrosine kinase (Syk) and fortreatment of a Syk-related inflammatory condition. Such conditionsinclude allergic conjunctivitis, ocular inflammation, dermatitis,rhinitis, asthma, allergy, or mast-cell disease, for example.

BACKGROUND OF THE INVENTION

Allergic conjunctivitis, ocular inflammation, dermatitis, rhinitis,asthma, allergy, and mast-cell disease have historically been treatedwith a regimen of oral, intranasal or topical antihistamines, or oral orintranasal steroids, or, in the case of allergy, allergen injectiontreatment. Systemic treatment typically requires higher concentrationsof the drug compound to be administered to afford an effectiveconcentration to reach the necessary treatment site. Antihistaminecompounds are known to have central nervous system activity; drowsinessand drying of mucus membranes are a common side-effect of antihistamineuse.

Signaling through immune receptors such as the IgE receptor (FcεRI)involves the recruitment and activation of multiple components of thesignaling cascade, including the non-receptor tyrosine kinase, Syk. Sykactivation leads to activation of the PLCγ and PI3K pathways andultimately to mast cell degranulation and activation. Targeting the SykmRNA would reduce the levels of Syk protein and interrupt the FcεRIpathway. This action would interfere with the IgE mediated mast celldegranulation and release of histamine and other pro-inflammatorymediators.

Inhibition of allergic inflammation in the airways using aerosolizedantisense to Syk kinase is reported by Stenton, G. R. et al., (JImmunol. 169:1028-1036 (2002)). Inhibition of Syk expression by means ofsmall interfering RNA (siRNA) in a bronchial epithelial cell line HS-24and in a rat model of ovalbumin (OA)-induced asthma reportedly inhibitedthe expression of hallmarks of induced inflammatory response (publishedinternational patent application WO 2005007623) Inhibition of expressionof genes involved in IgE production using a short hairpin RNA targetingFCεR1A and treatment of IgE-mediated diseases such as asthma andallergic rhinitis were reported in published international patentapplication WO 2005085443. None of these documents discloses theinterfering RNAs of the present invention.

Additional agents and treatment methods would be desirable for targetingthe Syk tyrosine kinase, thereby blocking the actions of endogenous mastcell degranulation, and release of histamine and other pro-inflammatorymediators while avoiding the side effects of systemic antihistaminetreatment. Embodiments of the present invention address the need in theart for such agents and treatment methods.

SUMMARY OF THE INVENTION

Embodiments of the present invention overcome these and other drawbacksof the prior art by providing highly potent and efficacious treatment,prevention or intervention of a Syk-related inflammatory condition. Inone aspect, methods of the invention include treating a subject having aSyk-related inflammatory condition or at risk of developing aSyk-related inflammatory condition by administering interfering RNAsthat silence expression of Syk mRNA, thus interfering with the PLCγ andPI3K signaling pathways and preventing a cascade of events related tomast cell degranulation, and release of histamine and otherpro-inflammatory mediators in a Syk-related inflammatory condition.

The present invention is directed to interfering RNAs that target SykmRNA and thereby interfere with Syk mRNA expression. The interferingRNAs of the invention are useful for treating patients with aSyk-related condition or at risk of developing a Syk-related condition.

An embodiment of the invention is a method of attenuating expression ofSyk mRNA of a subject, the method comprising administering to thesubject a composition comprising an effective amount of interfering RNAhaving a length of 19 to 49 nucleotides and a pharmaceuticallyacceptable carrier. The expression of Syk mRNA is attenuated thereby.

Another embodiment of the invention is a method of treating aSyk-related inflammatory condition in a subject in need thereof. Themethod comprises administering to the subject a composition comprisingan effective amount of interfering RNA having a length of 19 to 49nucleotides, and a pharmaceutically acceptable carrier. The Syk-relatedinflammatory condition is treated thereby. In one embodiment, thesubject is a human and the human has a Syk-related inflammatorycondition and, in another embodiment, the subject is a human and thehuman is at risk of developing a Syk-related inflammatory condition.

For the above cited embodiments, the interfering RNA comprises a regionof at least 13 contiguous nucleotides having at least 90% sequencecomplementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of an mRNA corresponding to anyone of SEQ ID NO:2, SEQ ID NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ IDNO:47.

In further embodiments of the above-cited methods, the compositionfurther comprises a second interfering RNA having a length of 19 to 49nucleotides and comprising a region of at least 13 contiguousnucleotides having at least 90% sequence complementarity to, or at least90% sequence identity with, the penultimate 13 nucleotides of the 3′ endof a second mRNA corresponding to any one of SEQ ID NO:2, SEQ IDNO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47.

In yet another embodiment of the invention, a method of attenuatingexpression of Syk mRNA of a subject comprises administering to thesubject a composition comprising an effective amount of interfering RNAhaving a length of 19 to 49 nucleotides and a pharmaceuticallyacceptable carrier and the interfering RNA comprises a sense nucleotidestrand, an antisense nucleotide strand, and a region of at leastnear-perfect contiguous complementarity of at least 19 nucleotides wherethe antisense strand hybridizes under physiological conditions to aportion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide 642,789, 791, 860, 861, 862, 867, 868, 1009, 1273, 1394, 1436, 1471, 1472,1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880, 1947, 2022, 2036, 2328,2329, 2473, 2509, 2520, 2524, 2558, or 2613. The expression of Syk mRNAis attenuated thereby. In a further embodiment, the compositioncomprises an effective amount of interfering RNA having a length of 19to 49 nucleotides where the antisense strand hybridizes underphysiological conditions to a portion of mRNA corresponding to SEQ IDNO:1 comprising nucleotide 1007, 1698, or 1769. In one embodiment, theantisense strand hybridizes to a portion of mRNA corresponding to SEQ IDNO:1 beginning with nucleotide 1007, 1698, or 1769 and is 19 or 20nucleotides long.

A method of treating a Syk-related inflammatory condition in a subjectin need thereof is an embodiment of the invention, the method comprisingadministering to the subject a composition comprising an effectiveamount of interfering RNA having a length of 19 to 49 nucleotides, and apharmaceutically acceptable carrier, the interfering RNA comprising asense nucleotide strand, an antisense nucleotide strand, and a region ofat least near-perfect contiguous complementarity of at least 19nucleotides; wherein the antisense strand hybridizes under physiologicalconditions to a portion of mRNA corresponding to SEQ ID NO:1 comprisingnucleotide 642, 789, 791, 860, 861, 862, 867, 868, 1009, 1273, 1394,1436, 1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880, 1947,2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524, 2558, or 2613. TheSyk-related inflammatory condition is treated thereby. In a furtherembodiment, the composition comprises an effective amount of interferingRNA having a length of 19 to 49 nucleotides where the antisense strandhybridizes under physiological conditions to a portion of mRNAcorresponding to SEQ ID NO:1 comprising nucleotide 1007, 1698, or 1769.In one embodiment, the antisense strand hybridizes to a portion of mRNAcorresponding to SEQ ID NO:1 beginning with nucleotide 1007, 1698, or1769 and is 19 or 20 nucleotides long.

A second interfering RNA having a length of 19 to 49 nucleotides couldalso be administered to the subject; the second interfering RNAcomprising a sense nucleotide strand, an antisense nucleotide strand,and a region of at least near-perfect complementarity of at least 19nucleotides wherein the antisense strand of the second interfering RNAhybridizes under physiological conditions to a second portion of mRNAcorresponding to SEQ ID NO:1 comprising nucleotide 642, 789, 791, 860,861, 862, 867, 868, 1009, 1273, 1394, 1436, 1471, 1472, 1533, 1535,1547, 1680, 1738, 1739, 1766, 1880, 1947, 2022, 2036, 2328, 2329, 2473,2509, 2520, 2524, 2558, or 2613. A second interfering RNA having alength of 19 to 49 nucleotides could be administered to the subject,where the antisense strand hybridizes under physiological conditions toa portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide1007, 1698, or 1769. In one embodiment, the antisense strand hybridizesto a portion of mRNA corresponding to SEQ ID NO:1 beginning withnucleotide 1007, 1698, or 1769 and is 19 or 20 nucleotides long.

A method of attenuating expression of Syk mRNA of a subject, comprisingadministering to the subject a composition comprising an effectiveamount of a single-stranded interfering RNA having a length of 19 to 49nucleotides, and a pharmaceutically acceptable carrier, where thesingle-stranded interfering RNA hybridizes under physiologicalconditions to a portion of mRNA corresponding to SEQ ID NO:1 comprisingthe nucleotides identified above is a further embodiment of theinvention.

The invention includes as a further embodiment a composition comprisingan interfering RNA having a length of 19 to 49 nucleotides, andcomprising a nucleotide sequence corresponding to any one of SEQ IDNO:2, SEQ ID NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47, or acomplement thereof; and a pharmaceutically acceptable carrier. Acomposition comprising an interfering RNA having a length of 19 to 49nucleotides, and comprising a nucleotide sequence corresponding to anyone of SEQ ID NO:41-SEQ ID NO:47, or a complement thereof; and apharmaceutically acceptable carrier is a further embodiment of theinvention.

A method of treating ocular inflammation or conjunctivitis in a subjectin need thereof is an embodiment of the invention. The method comprisesadministering to the subject a composition comprising a double strandedsiRNA molecule that down regulates expression of a Syk gene via RNAinterference, wherein each strand of the siRNA molecule is independentlyabout 19 to about 27 nucleotides in length; and one strand of the siRNAmolecule comprises a nucleotide sequence having substantialcomplementarity to an mRNA corresponding to the Syk gene so that thesiRNA molecule directs cleavage of the mRNA via RNA interference. In afurther embodiment of this method, each strand of the siRNA molecule isindependently about 19 nucleotides to about 25 nucleotides in length, orabout 19 nucleotides to about 21 nucleotides in length.

Use of any of the embodiments as described herein in the preparation ofa medicament for attenuating expression of Syk mRNA as set forth hereinis also an embodiment of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an SYK western blot of 293FT cells transfected with SYKsiRNAs #2, #5, #6, and #8, and a RISC-free control siRNA, each at 10 nM,1 nM, and 0.1 nM; a non-targeting control siRNA (NTC2) at 10 nM; and abuffer control (-siRNA). The arrows indicate the positions of the 72-kDaSYK and 42-kDa actin bands.

DETAILED DESCRIPTION OF THE INVENTION

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”.

The term “a Syk-related inflammatory condition” as used herein, includeshistamine and other pro-inflammatory mediated responses involved inconditions such as allergic conjunctivitis, ocular inflammation,dermatitis, rhinitis, asthma, allergy, and mast-cell disease, andincludes those cellular changes resulting from the expression ofSyk-mRNA that lead directly or indirectly to the Syk-relatedinflammatory condition. The interfering RNA provided herein provides forsuch silencing while avoiding undesirable side effects due tononspecific agents.

The term “allergic conjunctivitis,” as used herein, refers toinflammation of the conjunctiva which is the delicate membrane thatlines the eyelids and covers the exposed surface of the sclera. The term“allergic conjunctivitis” includes, for example, atopickeratoconjunctivitis, giant papillary conjunctivitis, hay feverconjunctivitis, perennial allergic conjunctivitis, and vernalkeratoconjunctivitis.

The term “dermatitis,” as used herein, refers to inflammation of theskin and includes, for example, allergic contact dermatitis, asteatoticdermatitis (dry skin on the lower legs), atopic dermatitis, cercarialdermatitis, contact dermatitis including irritant contact dermatitis andurushiol-induced contact dermatitis, dermatitis herpetiformis,dyshidrotic dermatitis, eczema, gravitational dermatitis, infectivedermatitis, nummular dermatitis, otitis externa, perioral dermatitis,Pseudomonas dermatitis (hot tub rash), and seborrhoeic dermatitis.

The term “rhinitis,” as used herein, refers to inflammation of themucous membranes of the nose and includes, for example, allergicrhinitis, atopic rhinitis, infectious rhinitis, irritant rhinitis,eosinophilic non-allergic rhinitis, rhinitis medicamentosa, andneutrophilic rhinosinusitis.

The term “asthma,” as used herein, refers to inflammation of the airpassages resulting in narrowing of the airways that transport air fromthe nose and mouth to the lungs and includes, for example, allergicasthma, atopic asthma, atopic bronchial IgE-mediated asthma, bronchialasthma, bronchiolytis, emphysematous asthma, essential asthma,exercise-induced asthma, extrinsic asthma caused by environmentalfactors, incipient asthma, intrinsic asthma caused by pathophysiologicdisturbances, non-allergic asthma, non-atopic asthma, and wheezy infantsyndrome.

The term “allergy,” as used herein, refers to an abnormal reaction ofthe immune system to a substance that is usually not harmful andincludes, for example, skin allergies such as atopic dermatitis, hives,and angioedema; respiratory allergies such as allergic rhinitis, andreactions to dust or mold; food allergies such as reactions to proteinsin cow's milk, egg whites, peanuts, wheat, soybeans, berries, shellfish,corn, beans, yellow food dye No. 5 and gum arabic; drug allergies suchas reactions to penicillin, sulfas, barbiturates, anticonvulsants,insulin, local anesthetics and contrast agents; and insect biteallergies such as reactions to venom in stings of bees, wasps, hornets,yellow jackets and fire ants.

The term “mast-cell disease,” as used herein, refers to systemicmastocytosis which is characterized by mast cell accumulation intissues. Such accumulation can affect organs such as bone marrow, skin,the GI tract, the liver, and the spleen. Physicians diagnose systemicmastocytosis by finding mast cells in parts of the body other than theskin and generally treat with antihistamines.

The phrase “a region of at least 13 contiguous nucleotides having atleast 90% sequence complementarity to, or at least 90% sequence identitywith, the penultimate 13 nucleotides of the 3′ end of an mRNAcorresponding to any one of (a sequence identifier)” allows a onenucleotide substitution. Two nucleotide substitutions (i.e., 11/13=85%identity/complementarity) are not included in such a phrase.

“Hybridization” refers to a process in which single-stranded nucleicacids with complementary or near-complementary base sequences interactto form hydrogen-bonded complexes called hybrids. Hybridizationreactions are sensitive and selective. In vitro, the specificity ofhybridization (i.e., stringency) is controlled by the concentrations ofsalt or formamide in prehybridization and hybridization solutions, forexample, and by the hybridization temperature; such procedures are wellknown in the art. In particular, stringency is increased by reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature.

As used herein, the term “percent identity” describes the percentage ofcontiguous nucleotides in a first nucleic acid molecule that is the sameas in a set of contiguous nucleotides of the same length in a secondnucleic acid molecule. The term “percent complementarity” describes thepercentage of contiguous nucleotides in a first nucleic acid moleculethat can base pair in the Watson-Crick sense with a set of contiguousnucleotides in a second nucleic acid molecule.

Attenuating expression of an mRNA: The phrase, “attenuating expressionof an mRNA,” as used herein, means administering or expressing an amountof interfering RNA (e.g., an siRNA) to reduce translation of the targetmRNA into protein, either through mRNA cleavage or through directinhibition of translation. The reduction in expression of the targetmRNA or the corresponding protein is commonly referred to as“knock-down” and is reported relative to levels present followingadministration or expression of a non-targeting control RNA (e.g., anon-targeting control siRNA). Knock-down of expression of an amountincluding and between 50% and 100% is contemplated by embodimentsherein. However, it is not necessary that such knock-down levels beachieved for purposes of the present invention. In one embodiment, asingle interfering RNA targeting Syk mRNA is administered. In otherembodiments, two or more interfering RNAs targeting Syk mRNA areadministered.

“Hybridization” refers to a process in which single-stranded nucleicacids with complementary or near-complementary base sequences interactto form hydrogen-bonded complexes called hybrids. Hybridizationreactions are sensitive and selective. In vitro, the specificity ofhybridization (i.e., stringency) is controlled by the concentrations ofsalt or formamide in prehybridization and hybridization solutions, forexample, and by the hybridization temperature; such procedures are wellknown in the art. In particular, stringency is increased by reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature.

For example, high stringency conditions could occur at about 50%formamide at 37° C. to 42° C. Reduced stringency conditions could occurat about 35% to 25% formamide at 30° C. to 35° C. Examples of stringencyconditions for hybridization are provided in Sambrook, J., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. Further examples of stringenthybridization conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing, orhybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC, or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The temperature for hybridization is about 5-10° C. less than themelting temperature (T_(m)) of the hybrid where T_(m) is determined forhybrids between 19 and 49 base pairs in length using the followingcalculation: T_(m)° C.=81.5+16.6(log₁₀ [Na+])+0.41 (% G+C)−(600/N) whereN is the number of bases in the hybrid, and [Na+] is the concentrationof sodium ions in the hybridization buffer.

Knock-down is commonly assessed by measuring the mRNA levels usingquantitative polymerase chain reaction (qPCR) amplification or bymeasuring protein levels by western blot or enzyme-linked immunosorbentassay (ELISA). Analyzing the protein level provides an assessment ofboth mRNA cleavage as well as translation inhibition. Further techniquesfor measuring knock-down include RNA solution hybridization, nucleaseprotection, northern hybridization, gene expression monitoring with amicroarray, antibody binding, radioimmunoassay, and fluorescenceactivated cell analysis.

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. While not wanting to bebound by theory, RNAi begins with the cleavage of longer dsRNAs intosmall interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer.SiRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to25 nucleotides, or 21 to 22 nucleotides in length and often contain2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. Onestrand of the siRNA is incorporated into a ribonucleoprotein complexknown as the RNA-induced silencing complex (RISC). RISC uses this siRNAstrand to identify mRNA molecules that are at least partiallycomplementary to the incorporated siRNA strand, and then cleaves thesetarget mRNAs or inhibits their translation. Therefore, the siRNA strandthat is incorporated into RISC is known as the guide strand or theantisense strand. The other siRNA strand, known as the passenger strandor the sense strand, is eliminated from the siRNA and is at leastpartially homologous to the target mRNA. Those of skill in the art willrecognize that, in principle, either strand of an siRNA can beincorporated into RISC and function as a guide strand. However, siRNAdesign (e.g., decreased siRNA duplex stability at the 5′ end of theantisense strand) can favor incorporation of the antisense strand intoRISC.

RISC-mediated cleavage of mRNAs having a sequence at least partiallycomplementary to the guide strand leads to a decrease in the steadystate level of that mRNA and of the corresponding protein encoded bythis mRNA. Alternatively, RISC can also decrease expression of thecorresponding protein via translational repression without cleavage ofthe target mRNA. Other RNA molecules and RNA-like molecules can alsointeract with RISC and silence gene expression. Examples of other RNAmolecules that can interact with RISC include short hairpin RNAs(shRNAs), single-stranded siRNAs, microRNAs (miRNAs), anddicer-substrate 27-mer duplexes. The term “siRNA” as used herein refersto a double-stranded interfering RNA unless otherwise noted. Examples ofRNA-like molecules that can interact with RISC include RNA moleculescontaining one or more chemically modified nucleotides, one or moredeoxyribonucleotides, and/or one or more non-phosphodiester linkages.For purposes of the present discussion, all RNA or RNA-like moleculesthat can interact with RISC and participate in RISC-mediated changes ingene expression will be referred to as “interfering RNAs.” SiRNAs,shRNAs, miRNAs, and dicer-substrate 27-mer duplexes are, therefore,subsets of “interfering RNAs.”

Interfering RNA of embodiments of the invention appear to act in acatalytic manner for cleavage of target mRNA, i.e., interfering RNA isable to effect inhibition of target mRNA in substoichiometric amounts.As compared to antisense therapies, significantly less interfering RNAis required to provide a therapeutic effect under such cleavageconditions.

The present invention relates to the use of interfering RNA to inhibitthe expression of Syk mRNA, a non-receptor tyrosine kinase. Signalingthrough immune receptors such as the IgE receptor (FcεRI) involves therecruitment and activation of multiple components of the signalingcascade, including Syk. Syk activation leads to activation of the PLCγand PI3K pathways and ultimately to mast cell degranulation andactivation. Targeting the Syk mRNA as provided herein reduces the levelof Syk protein and interrupts the FcεRI pathway. This action interfereswith the IgE mediated mast cell degranulation and release of histamineand other pro-inflammatory mediators. According to the presentinvention, interfering RNAs provided exogenously or expressedendogenously are particularly effective at silencing Syk mRNA.

Nucleic acid sequences cited herein are written in a 5′ to 3′ directionunless indicated otherwise. The term “nucleic acid,” as used herein,refers to either DNA or RNA or a modified form thereof comprising thepurine or pyrimidine bases present in DNA (adenine “A,” cytosine “C,”guanine “G,” thymine “T”) or in RNA (adenine “A,” cytosine “C,” guanine“G,” uracil “U”). Interfering RNAs provided herein may comprise “T”bases, particularly at 3′ ends, even though “T” bases do not naturallyoccur in RNA. “Nucleic acid” includes the terms “oligonucleotide” and“polynucleotide” and can refer to a single-stranded molecule or adouble-stranded molecule. A double-stranded molecule is formed byWatson-Crick base pairing between A and T bases, C and G bases, andbetween A and U bases. The strands of a double-stranded molecule mayhave partial, substantial or full complementarity to each other and willform a duplex hybrid, the strength of bonding of which is dependent uponthe nature and degree of complementarity of the sequence of bases.

An mRNA sequence is readily deduced from the sequence of thecorresponding DNA sequence. For example, SEQ ID NO:1 provides the sensestrand sequence of DNA corresponding to the mRNA for Syk. The mRNAsequence is identical to the DNA sense strand sequence with the “T”bases replaced with “U” bases. Therefore, the mRNA sequence of Syk isknown from SEQ ID NO:1.

Spleen Tyrosine Kinase (Syk) mRNA: The GenBank database provides the DNAsequence for Syk as accession no. NM_(—)003177, provided in the“Sequence Listing” as SEQ ID NO:1. SEQ ID NO:1 provides the sense strandsequence of DNA that corresponds to the mRNA encoding Syk (with theexception of “T” bases for “U” bases). The coding sequence for Syk isfrom nucleotides 148-2055.

Equivalents of the above cited Syk mRNA sequence are alternative spliceforms, allelic forms, isozymes, or a cognate thereof. A cognate is aspleen tyrosine kinase mRNA from another mammalian species that ishomologous to SEQ ID NO:1 (i.e., an ortholog). Syk nucleic acidsequences related to SEQ ID NO:1 include those having GenBank accessionnumbers BC001645, BC002962, L28824, X73568, BC011399, and Z29630.

Inhibition of Syk may also be determined in vitro by monitoringhistamine release in immunologically-challenged mast cells as follows.Mono-dispersed human mast cell preparations release pro-inflammatorymediators upon immunological activation. The degree of activation isdetermined by quantification of histamine released into culturesupernatant upon stimulation of the cells with antigen or anti-humanIgE. (See Miller et al. Ocular Immunol Inflamm. 4:39-49, 2006).Immunological challenge of mast cells transfected with interfering RNAtargeting Syk results in significantly less histamine release thanobserved with immunological challenge of non-transfected mast cells ormast cells transfected with a non-targeting control interfering RNA.

The inhibition of Syk may also be determined by examining IgE mediatedrelease of histamine in vitro using human conjunctival tissue mast cellsor in LAD2 mast cells. Human conjunctival tissue mast cells are obtainedusing the methodology outlined in U.S. Pat. No. 5,360,720, for example.LAD2 mast cells are licensed from the Laboratory of Allergic Diseases,National Institutes of Health, Bethesda, Md. (Leuk Res. 2003 Aug.27(8):677-82). Briefly, human conjunctival tissue mast cells areenzymatically released from human conjunctival tissues and thenpartially enriched by density centrifugation over a PERCOLL® cushion. Amonodispersed cell suspension is obtained from the resulting pellet, andthese cells are used for a histamine release assay. Cells aretransfected with a Syk interfering RNA or a control interfering RNA 72 hprior to anti-human IgE stimulation, which triggers mast celldegranulation and histamine release to the supernatant. Histamine isthen measured in the supernatant by RIA (Beckman Coulter), EIA (BeckmanCoulter) or other method known to one of skill in the art. A decrease inhistamine release in Syk interfering RNA transfected cells relative tocontrol transfected cells or non-transfected cells indicates that aninterfering RNA is effective at attenuating Syk and interrupting theFceRI signaling pathway.

The LAD2 mast cell line is used in much the same way with the exceptionthat the cells are passively sensitized with human IgE myeloma prior tostimulation with anti-human IgE to cross-link receptor bound IgE andtrigger degranulation.

Inhibition of Syk is also inferred in a human or mammal by observing animprovement in a Syk-related condition symptom such as improvement insymptoms related to allergic conjunctivitis, ocular inflammation,dermatitis, rhinitis, asthma, allergy, or mast-cell disease. Improvementin any of edema, itching, inflammation, or tolerance to environmentalchallenges, for example, is indicative of inhibition of Syk.

Interfering RNA: In one embodiment of the invention, interfering RNA(e.g., siRNA) has a sense strand and an antisense strand, and the senseand antisense strands comprise a region of at least near-perfectcontiguous complementarity of at least 19 nucleotides. In a furtherembodiment of the invention, interfering RNA (e.g., siRNA) has a sensestrand and an antisense strand, and the antisense strand comprises aregion of at least near-perfect contiguous complementarity of at least19 nucleotides to a target sequence of Syk mRNA, and the sense strandcomprises a region of at least near-perfect contiguous identity of atleast 19 nucleotides with a target sequence of Syk mRNA. In a furtherembodiment of the invention, the interfering RNA comprises a region ofat least 13, 14, 15, 16, 17, or 18 contiguous nucleotides havingpercentages of sequence complementarity to or, having percentages ofsequence identity with, the penultimate 13, 14, 15, 16, 17, or 18nucleotides, respectively, of the 3′ end of the corresponding targetsequence within an mRNA.

The length of each strand of the interfering RNA comprises 19 to 49nucleotides, and may comprise a length of 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, or 49 nucleotides.

The antisense strand of an siRNA is the active guiding agent of thesiRNA in that the antisense strand is incorporated into RISC, thusallowing RISC to identify target mRNAs with at least partialcomplementarity to the antisense siRNA strand for cleavage ortranslational repression.

In embodiments of the present invention, interfering RNA targetsequences (e.g., siRNA target sequences) within a target mRNA sequenceare selected using available design tools. Interfering RNAscorresponding to a Syk target sequence are then tested by transfectionof cells expressing the target mRNA followed by assessment of knockdownas described above.

Techniques for selecting target sequences for siRNAs are provided byTuschl, T. et al., “The siRNA User Guide,” revised May 6, 2004,available on the Rockefeller University web site; by Technical Bulletin#506, “siRNA Design Guidelines,” Ambion Inc. at Ambion's web site; andby other web-based design tools at, for example, the Invitrogen,Dharmacon, Integrated DNA Technologies, Genscript, or Proligo web sites.Initial search parameters can include G/C contents between 35% and 55%and siRNA lengths between 19 and 27 nucleotides. The target sequence maybe located in the coding region or in the 5′ or 3′ untranslated regionsof the mRNAs.

An embodiment of a 19-nucleotide DNA target sequence for Syk mRNA ispresent at nucleotides 789 to 807 of SEQ ID NO:1:

5′- GCACTATCGCATCGACAAA -3′. SEQ ID NO: 2An siRNA of the invention for targeting a corresponding mRNA sequence ofSEQ ID NO:2 and having 21-nucleotide strands and a 2-nucleotide 3′overhang is:

  5′- GCACUAUCGCAUCGACAAANN -3′ SEQ ID NO: 3 3′-NNCGUGAUAGCGUAGCUGUUU -5′. SEQ ID NO: 4Each “N” residue can be any nucleotide (A, C, G, U, T) or modifiednucleotide. The 3′ end can have a number of “N” residues between andincluding 1, 2, 3, 4, 5, and 6. The “N” residues on either strand can bethe same residue (e.g., UU, AA, CC, GG, or TT) or they can be different(e.g., AC, AG, AU, CA, CG, CU, GA, GC, GU, UA, UC, or UG). The 3′overhangs can be the same or they can be different. In one embodiment,both strands have a 3′UU overhang.

An siRNA of the invention for targeting a corresponding mRNA sequence ofSEQ ID NO:2 and having 21-nucleotide strands and a 3′UU overhang on eachstrand is:

  5′- GCACUAUCGCAUCGACAAAUU -3′ SEQ ID NO: 53′- UUCGUGAUAGCGUAGCUGUUU -5′. SEQ ID NO: 6

The interfering RNA may also have a 5′ overhang of nucleotides or it mayhave blunt ends. An siRNA of the invention for targeting a correspondingmRNA sequence of SEQ ID NO:2 and having 19-nucleotide strands and bluntends is:

5′- GCACUAUCGCAUCGACAAA -3′ SEQ ID NO: 7 3′- CGUGAUAGCGUAGCUGUUU -5′.SEQ ID NO: 8

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA). AnshRNA of the invention targeting a corresponding mRNA sequence of SEQ IDNO:2 and having a 19 bp double-stranded stem region and a 3′UU overhangis:

N is a nucleotide A, T, C, G, U, or a modified form known by one ofordinary skill in the art. The number of nucleotides N in the loop is anumber between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9,or 9 to 11, or the number of nucleotides N is 9. Some of the nucleotidesin the loop can be involved in base-pair interactions with othernucleotides in the loop. Examples of oligonucleotide sequences that canbe used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. etal. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al.(2002) RNA 8:1454). It will be recognized by one of skill in the artthat the resulting single chain oligonucleotide forms a stem-loop orhairpin structure comprising a double-stranded region capable ofinteracting with the RNAi machinery.

The siRNA target sequence identified above can be extended at the 3′ endto facilitate the design of dicer-substrate 27-mer duplexes. Extensionof the 19-nucleotide DNA target sequence (SEQ ID NO:2) identified in theSyk DNA sequence (SEQ ID NO:1) by 6 nucleotides yields a 25-nucleotideDNA target sequence present at nucleotides 789 to 813 of SEQ ID NO:1:

5'- GCACTATCGCATCGACAAAGACAAG -3'. SEQ ID NO: 10A dicer-substrate 27-mer duplex of the invention for targeting acorresponding mRNA sequence of SEQ ID NO:10 is:

  5′- GCACUAUCGCAUCGACAAAGACAAG -3′ SEQ ID NO: 113′- UUCGUGAUAGCGUAGCUGUUUCUGUUC -5′. SEQ ID NO: 12The two nucleotides at the 3′ end of the sense strand (i.e., the AGnucleotides of SEQ ID NO:11) may be deoxynucleotides for enhancedprocessing. Design of dicer-substrate 27-mer duplexes from 19-21nucleotide target sequences, such as provided herein, is furtherdiscussed by the Integrated DNA Technologies (IDT) website and by Kim,D.-H. et al., (February, 2005) Nature Biotechnology 23:2; 222-226.

When interfering RNAs are produced by chemical synthesis,phosphorylation at the 5′ position of the nucleotide at the 5′ end ofone or both strands (when present) can enhance siRNA efficacy andspecificity of the bound RISC complex but is not required sincephosphorylation can occur intracellularly.

Also disclosed herein is a method of attenuating expression of Syk mRNAof a subject, comprising:

-   -   administering to the subject a composition comprising an        effective amount of interfering RNA (iRNA) having a length of 19        to 49 nucleotides and a pharmaceutically acceptable carrier, the        interfering RNA comprising a region selected from the group        consisting of:        -   a region of at least 13 contiguous nucleotides having at            least 90% sequence complementarity to, or at least 90%            sequence identity with, the penultimate 13 nucleotides of            the 3′ end of an mRNA corresponding to any one of SEQ ID            NO:2 and SEQ ID NO:13-SEQ ID NO:40;        -   a region of at least 14 contiguous nucleotides having at            least 85% sequence complementarity to, or at least 85%            sequence identity with, the penultimate 14 nucleotides of            the 3′ end of an mRNA corresponding to any one of SEQ ID            NO:2, SEQ ID NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID            NO:47; and,        -   a region of at least 15, 16, 17, or 18 contiguous            nucleotides having at least 80% sequence complementarity to,            or at least 80% sequence identity with, the penultimate 15,            16, 17, or 18 nucleotides, respectively, of the 3′ end of an            mRNA corresponding to any one of SEQ ID NO:2, SEQ ID            NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47,        -   wherein the expression of Syk mRNA is attenuated thereby.

Also disclosed is a method of treating a Syk-related inflammatorycondition in a subject in need thereof, comprising:

-   -   administering to the subject a composition comprising an        effective amount of interfering RNA having a length of 19 to 49        nucleotides, and a pharmaceutically acceptable carrier, wherein        -   the interfering RNA comprises a region selected from the            group consisting of a region of at least 13 contiguous            nucleotides having at least 90% sequence complementarity to,            or at least 90% sequence identity with, the penultimate 13            nucleotides of the 3′ end of an mRNA corresponding to any            one of SEQ ID NO:2, SEQ ID NO:13-SEQ ID NO:40, and SEQ ID            NO:44-SEQ ID NO:47;        -   a region of at least 14 contiguous nucleotides having at            least 85% sequence complementarity to, or at least 85%            sequence identity with, the penultimate 14 nucleotides of            the 3′ end of an mRNA corresponding to any one of SEQ ID            NO:2 and SEQ ID NO:13-SEQ ID NO:40; and,        -   a region of at least 15, 16, 17, or 18 contiguous            nucleotides having at least 80% sequence complementarity to,            or at least 80% sequence identity with, the penultimate 15,            16, 17, or 18 nucleotides, respectively, of the 3′ end of an            mRNA corresponding to any one of SEQ ID NO:2, SEQ ID            NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47,    -   wherein the Syk-related inflammatory condition is treated        thereby.

Further embodiments comprise a method of attenuating expression of SykmRNA of a subject, comprising:

-   -   administering to the subject a composition comprising an        effective amount of interfering RNA having a length of 19 to 49        nucleotides and a pharmaceutically acceptable carrier, the        interfering RNA comprising:        -   a sense nucleotide strand, an antisense nucleotide strand,            and a region of at least near-perfect contiguous            complementarity of at least 19 nucleotides;        -   wherein the antisense strand hybridizes under physiological            conditions to a portion of mRNA selected from the group            consisting of        -   mRNA corresponding to SEQ ID NO:1 comprising nucleotide 642,            789, 791, 860, 861, 862, 867, 868, 1009, 1273, 1394, 1436,            1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880,            1947, 2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524, 2558,            or 2613, and,        -   mRNA corresponding to SEQ ID NO:1 comprising nucleotide            1007, 1698, or 1769,    -   wherein the expression of Syk mRNA is attenuated thereby.

Also disclosed herein are methods of treating a Syk-related inflammatorycondition in a subject in need thereof, comprising:

-   -   administering to the subject a composition comprising an        effective amount of interfering RNA having a length of 19 to 49        nucleotides, and a pharmaceutically acceptable carrier, the        interfering RNA comprising a sense nucleotide strand, an        antisense nucleotide strand, and a region of at least        near-perfect contiguous complementarity of at least 19        nucleotides;        -   wherein the antisense strand hybridizes under physiological            conditions to a portion of mRNA selected from the group            consisting of        -   mRNA corresponding to SEQ ID NO:1 comprising nucleotide 642,            789, 791, 860, 861, 862, 867, 868, 1009, 1273, 1394, 1436,            1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880,            1947, 2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524, 2558,            or 2613, and,        -   mRNA corresponding to SEQ ID NO:1 comprising nucleotide            1007, 1698, or 1769,    -   wherein the Syk-related inflammatory condition is treated        thereby.

Further embodiments comprise a method of attenuating expression of SykmRNA of a subject, comprising:

-   -   administering to the subject a composition comprising an        effective amount of a single-stranded interfering RNA having a        length of 19 to 49 nucleotides, and a pharmaceutically        acceptable carrier,        -   wherein the single-stranded interfering RNA hybridizes under            physiological conditions to a portion of mRNA selected from            the group consisting of        -   mRNA corresponding to SEQ ID NO:1 comprising nucleotide 642,            789, 791, 860, 861, 862, 867, 868, 1009, 1273, 1394, 1436,            1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880,            1947, 2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524, 2558,            or 2613, and,        -   mRNA corresponding to SEQ ID NO:1 comprising nucleotide            1007, 1698, or 1769,        -   and the interfering RNA has a region of at least            near-perfect contiguous complementarity with the hybridizing            portion of mRNA corresponding to SEQ ID NO:1,    -   wherein the expression of Syk mRNA is thereby attenuated.

Also disclosed are method of treating ocular inflammation orconjunctivitis in a subject in need thereof, comprising:

-   -   administering to the subject a composition comprising a double        stranded siRNA molecule that down regulates expression of a Syk        gene via RNA interference,    -   wherein:    -   each strand of the siRNA molecule is independently about 19 to        about 27 nucleotides in length; and    -   one strand of the siRNA molecule comprises a nucleotide sequence        having substantial complementarity to an mRNA corresponding to        the Syk gene so that the siRNA molecule directs cleavage of the        mRNA via RNA interference.

Also disclosed are various compositions comprising an interfering RNAhaving a length of 19 to 49 nucleotides, and comprising a nucleotidesequence corresponding to any one of SEQ ID NO:2, SEQ ID NO:13-SEQ IDNO:40, and SEQ ID NO:44-SEQ ID NO:47, or a complement thereof; and apharmaceutically acceptable carrier. Various other compositions comprisean interfering RNA having a length of 19 to 49 nucleotides, andconsisting essentially of a nucleotide sequence corresponding to any oneof SEQ ID NO:41-SEQ ID NO:47, or a complement thereof; and apharmaceutically acceptable carrier.

While a particular embodiment of the invention has been shown anddescribed, numerous variations and alternate embodiments will occur tothose skilled in the art. Accordingly, it is intended that the inventionbe limited only in terms of the appended claims.

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changes tothe claims that come within the meaning and range of equivalency of theclaims are to be embraced within their scope. Further, all publisheddocuments, patents, and applications mentioned herein are herebyincorporated by reference, as if presented in their entirety.

Examples

Table 1 lists examples of Syk DNA target sequences of SEQ ID NO:1 fromwhich siRNAs of the present invention are designed in a manner as setforth above. Syk encodes spleen tyrosine kinase, as noted above.

TABLE 1 Syk Target Sequences for siRNAs # of Starting    Nucleotide withSEQ Syk Target reference to ID Sequence SEQ ID NO: 1 NO:GCACTATCGCATCGACAAA 789 2 ACTATCGCATCGACAAAGA 791 13 GGCAGCTAGTCGAGCATTA860 14 GCAGCTAGTCGAGCATTAT 861 15 CAGCTAGTCGAGCATTATT 862 16AGTCGAGCATTATTCTTAT 867 17 GTCGAGCATTATTCTTATA 868 18AGTTATTAGCAGAAGCAAA 1394 19 CGTACATCGTGCGCATGAT 1436 20GAGTCCTGGATGCTAGTTA 1471 21 AGTCCTGGATGCTAGTTAT 1472 22ACAGACATGTCAAGGATAA 1535 23 AGGATAAGAACATCATAGA 1547 24TGATTTCGGACTCTCCAAA 1680 25 CATGGAAAGTGGCCTGTCA 1738 26ATGGAAAGTGGCCTGTCAA 1739 27 CTCCGGAATGCATCAACTA 1766 28GAAGTGAAGTCACCGCTAT 1880 29 GATGTACGATCTCATGAAT 1947 30GCTGCGCAATTACTACTAT 2022 31 ACTATGACGTGGTGAACTA 2036 32TTACGATCTGTTTCCAAAT 2328 33 TACGATCTGTTTCCAAATC 2329 34CTTAGCATGTGACTCCTGA 2473 35 AGGAATTTGGCTGCTTCTA 2509 36TGCTTCTACGGCCATGAGA 2520 37 TCTACGGCCATGAGACTGA 2524 38AAGCTTTCCTGACAATAAA 2558 39 CCAGTGCAGTTTCTAAGCA 2613 40

TABLE 2 Further Syk Target Sequences for siRNAs # of Starting   Nucleotide with SEQ Syk Target  reference to ID Sequence SEQ ID NO: 1NO: GTGGAATAATCTCAAGAAT 1007 41 AGCACTGCGTGCTGATGAA 1698 42CGGAATGCATCAACTACTA 1769 43 AAUGCCUUGGUUCCAUGGA 642 44GGAAUAAUCUCAAGAAUCA 1009 45 GAACUGGGCUCUGGUAAUU 1273 46GAACAGACAUGUCAAGGAU 1533 47

As cited in the examples above, one of skill in the art is able to usethe target sequence information provided in Table 1 to designinterfering RNAs having a length shorter or longer than the sequencesprovided in Table 1 by referring to the sequence position in SEQ ID NO:1and adding or deleting nucleotides complementary or near complementaryto SEQ ID NO:1.

The target RNA cleavage reaction guided by siRNAs and other forms ofinterfering RNA is highly sequence specific. In general, siRNAcontaining a sense nucleotide strand identical in sequence to a portionof the target mRNA and an antisense nucleotide strand exactlycomplementary to a portion of the target mRNA are siRNA embodiments forinhibition of mRNAs cited herein. However, 100% sequence complementaritybetween the antisense siRNA strand and the target mRNA, or between theantisense siRNA strand and the sense siRNA strand, is not required topractice the present invention. Thus, for example, the invention allowsfor sequence variations that might be expected due to genetic mutation,strain polymorphism, or evolutionary divergence.

In one embodiment of the invention, the antisense strand of the siRNAhas at least near-perfect contiguous complementarity of at least 19nucleotides with the target mRNA. “Near-perfect,” as used herein, meansthe antisense strand of the siRNA is “substantially complementary to,”and the sense strand of the siRNA is “substantially identical” to atleast a portion of the target mRNA. “Identity,” as known by one ofordinary skill in the art, is the degree of sequence relatedness betweennucleotide sequences as determined by matching the order and identity ofnucleotides between the sequences. In one embodiment, the antisensestrand of an siRNA having 80% and between 80% up to 100%complementarity, for example, 85%, 90% or 95% complementarity, to thetarget mRNA sequence are considered near-perfect complementarity and maybe used in the present invention. “Perfect” contiguous complementarityis standard Watson-Crick base pairing of adjacent base pairs. “At leastnear-perfect” contiguous complementarity includes “perfect”complementarity as used herein. Computer methods for determiningidentity or complementarity are designed to identify the greatest degreeof matching of nucleotide sequences, for example, BLASTN (Altschul, S.F., et al. (1990) J. Mol. Biol. 215:403-410).

The relationship between a target mRNA (sense strand) and one strand ofan siRNA (the sense strand) is that of identity. The sense strand of ansiRNA is also called a passenger strand, if present. The relationshipbetween a target mRNA (sense strand) and the other strand of an siRNA(the antisense strand) is that of complementarity. The antisense strandof an siRNA is also called a guide strand.

The penultimate base in a nucleic acid sequence that is written in a 5′to 3′ direction is the next to the last base, i.e., the base next to the3′ base. The penultimate 13 bases of a nucleic acid sequence written ina 5′ to 3′ direction are the last 13 bases of a sequence next to the 3′base and not including the 3′ base. Similarly, the penultimate 14, 15,16, 17, or 18 bases of a nucleic acid sequence written in a 5′ to 3′direction are the last 14, 15, 16, 17, or 18 bases of a sequence,respectively, next to the 3′ base and not including the 3′ base.

In one embodiment of the invention, the region of contiguous nucleotidesis a region of at least 14 contiguous nucleotides having at least 85%sequence complementarity to, or at least 85% sequence identity with, thepenultimate 14 nucleotides of the 3′ end of an mRNA corresponding to thesequence identified by each sequence identifier. Two nucleotidesubstitutions (i.e., 12/14=86% identity/complementarity) are included insuch a phrase.

In a further embodiment of the invention, the region of contiguousnucleotides is a region of at least 15, 16, 17, or 18 contiguousnucleotides having at least 80% sequence complementarity to, or at least80% sequence identity with, the penultimate 14 nucleotides of the 3′ endof an mRNA corresponding to the sequence of the sequence identifier.Three nucleotide substitutions are included in such a phrase.

The target sequence in the mRNAs corresponding to SEQ ID NO:1 may be inthe 5′ or 3′ untranslated regions of the mRNA as well as in the codingregion of the mRNA.

One or both of the strands of double-stranded interfering RNA may have a3′ overhang of from 1 to 6 nucleotides, which may be ribonucleotides ordeoxyribonucleotides or a mixture thereof. The nucleotides of theoverhang are not base-paired. In one embodiment of the invention, theinterfering RNA comprises a 3′ overhang of TT or UU. In anotherembodiment of the invention, the interfering RNA comprises at least oneblunt end. The termini usually have a 5′ phosphate group or a 3′hydroxyl group. In other embodiments, the antisense strand has a 5′phosphate group, and the sense strand has a 5′ hydroxyl group. In stillother embodiments, the termini are further modified by covalent additionof other molecules or functional groups.

The sense and antisense strands of the double-stranded siRNA may be in aduplex formation of two single strands as described above or may be asingle molecule where the regions of complementarity are base-paired andare covalently linked by a hairpin loop so as to form a single strand.It is believed that the hairpin is cleaved intracellularly by a proteintermed dicer to form an interfering RNA of two individual base-pairedRNA molecules.

Interfering RNAs may differ from naturally-occurring RNA by theaddition, deletion, substitution or modification of one or morenucleotides. Non-nucleotide material may be bound to the interferingRNA, either at the 5′ end, the 3′ end, or internally. Such modificationsare commonly designed to increase the nuclease resistance of theinterfering RNAs, to improve cellular uptake, to enhance cellulartargeting, to assist in tracing the interfering RNA, to further improvestability, or to reduce the potential for activation of the interferonpathway. For example, interfering RNAs may comprise a purine nucleotideat the ends of overhangs. Conjugation of cholesterol to the 3′ end ofthe sense strand of an siRNA molecule by means of a pyrrolidine linker,for example, also provides stability to an siRNA.

Further modifications include a 3′ terminal biotin molecule, a peptideknown to have cell-penetrating properties, a nanoparticle, apeptidomimetic, a fluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugarportion, or on the phosphate portion of the molecule and function inembodiments of the present invention. Modifications includesubstitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiolgroups, or a combination thereof, for example. Nucleotides may besubstituted with analogs with greater stability such as replacing aribonucleotide with a deoxyribonucleotide, or having sugar modificationssuch as 2′ OH groups replaced by 2′ amino groups, 2′ O-methyl groups, 2′methoxyethyl groups, or a 2′-O, 4′-C methylene bridge, for example.Examples of a purine or pyrimidine analog of nucleotides include axanthine, a hypoxanthine, an azapurine, a methylthioadenine,7-deaza-adenosine and O- and N-modified nucleotides. The phosphate groupof the nucleotide may be modified by substituting one or more of theoxygens of the phosphate group with nitrogen or with sulfur(phosphorothioates). Modifications are useful, for example, to enhancefunction, to improve stability or permeability, or to directlocalization or targeting.

There may be a region or regions of the antisense interfering RNA strandthat is (are) not complementary to a portion of SEQ ID NO:1.Non-complementary regions may be at the 3′, 5′ or both ends of acomplementary region or between two complementary regions.

Interfering RNAs may be generated exogenously by chemical synthesis, byin vitro transcription, or by cleavage of longer double-stranded RNAwith dicer or another appropriate nuclease with similar activity.Chemically synthesized interfering RNAs, produced from protectedribonucleoside phosphoramidites using a conventional DNA/RNAsynthesizer, may be obtained from commercial suppliers such as AmbionInc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon(Lafayette, Colo.). Interfering RNAs are purified by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof, for example. Alternatively, interfering RNA may beused with little if any purification to avoid losses due to sampleprocessing.

Interfering RNAs can also be expressed endogenously from plasmid orviral expression vectors or from minimal expression cassettes, forexample, PCR generated fragments comprising one or more promoters and anappropriate template or templates for the interfering RNA. Examples ofcommercially available plasmid-based expression vectors for shRNAinclude members of the pSilencer series (Ambion, Austin, Tex.) andpCpG-siRNA (InvivoGen, San Diego, Calif.). Viral vectors for expressionof interfering RNA may be derived from a variety of viruses includingadenovirus, adeno-associated virus, lentivirus (e.g., HIV, FIV, andEIAV), and herpes virus. Examples of commercially available viralvectors for shRNA expression include pSilencer adeno (Ambion, Austin,Tex.) and pLenti6/BLOCK-iT™-DEST (Invitrogen, Carlsbad, Calif.).Selection of viral vectors, methods for expressing the interfering RNAfrom the vector and methods of delivering the viral vector are withinthe ordinary skill of one in the art. Examples of kits for production ofPCR-generated shRNA expression cassettes include Silencer Express(Ambion, Austin, Tex.) and siXpress (Mirus, Madison, Wis.). A firstinterfering RNA may be administered via in vivo expression from a firstexpression vector capable of expressing the first interfering RNA and asecond interfering RNA may be administered via in vivo expression from asecond expression vector capable of expressing the second interferingRNA, or both interfering RNAs may be administered via in vivo expressionfrom a single expression vector capable of expressing both interferingRNAs.

Interfering RNAs may be expressed from a variety of eukaryotic promotersknown to those of ordinary skill in the art, including pol IIIpromoters, such as the U6 or H1 promoters, or pol II promoters, such asthe cytomegalovirus promoter. Those of skill in the art will recognizethat these promoters can also be adapted to allow inducible expressionof the interfering RNA.

Hybridization under Physiological Conditions: In certain embodiments ofthe present invention, an antisense strand of an interfering RNAhybridizes with an mRNA in vivo as part of the RISC complex.

The above-described in vitro hybridization assay provides a method ofpredicting whether binding between a candidate siRNA and a target willhave specificity. However, in the context of the RISC complex, specificcleavage of a target can also occur with an antisense strand that doesnot demonstrate high stringency for hybridization in vitro.

Single-stranded interfering RNA: As cited above, interfering RNAsultimately function as single strands. Single-stranded (ss) interferingRNA has been found to effect mRNA silencing, albeit less efficientlythan double-stranded RNA. Therefore, embodiments of the presentinvention also provide for administration of a ss interfering RNA thathybridizes under physiological conditions to a portion of SEQ ID NO:1and has a region of at least near-perfect contiguous complementarity ofat least 19 nucleotides with the hybridizing portion of SEQ ID NO:1. Thess interfering RNA of Table 1 has a length of 19 to 49 nucleotides asfor the ds interfering RNA cited above. The ss interfering RNA has a 5′phosphate or is phosphorylated in situ or in vivo at the 5′ position.The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the sugar (e.g., ribose,deoxyribose, or an analog of same) at the 5′ end of the polynucleotideor oligonucleotide.

SS interfering RNAs are synthesized chemically or by in vitrotranscription or expressed endogenously from vectors or expressioncassettes as for ds interfering RNAs. 5′ Phosphate groups may be addedvia a kinase, or a 5′ phosphate may be the result of nuclease cleavageof an RNA. Delivery is as for ds interfering RNAs. In one embodiment, ssinterfering RNAs having protected ends and nuclease resistantmodifications are administered for silencing. SS interfering RNAs may bedried for storage or dissolved in an aqueous solution. The solution maycontain buffers or salts to inhibit annealing or for stabilization.

Hairpin interfering RNA: A hairpin interfering RNA is a single molecule(e.g., a single oligonucleotide chain) that comprises both the sense andantisense strands of an interfering RNA in a stem-loop or hairpinstructure (e.g., a shRNA). For example, shRNAs can be expressed from DNAvectors in which the DNA oligonucleotides encoding a sense interferingRNA strand are linked to the DNA oligonucleotides encoding the reversecomplementary antisense interfering RNA strand by a short spacer. Ifneeded for the chosen expression vector, 3′ terminal T's and nucleotidesforming restriction sites may be added. The resulting RNA transcriptfolds back onto itself to form a stem-loop structure.

Mode of administration: Interfering RNA may be delivered via aerosol,buccal, dermal, intradermal, inhaling, intramuscular, intranasal,intraocular, intrapulmonary, intravenous, intraperitoneal, nasal,ocular, oral, otic, parenteral, patch, subcutaneous, sublingual,topical, or transdermal administration, for example.

Administration may be directly to the eye by ocular tissueadministration such as periocular, conjunctival, subtenon, intracameral,intravitreal, intraocular, subretinal, subconjunctival, retrobulbar,intracanalicular, or suprachoroidal administration; by injection, bydirect application to the eye using a catheter or other placement devicesuch as a retinal pellet, intraocular insert, suppository or an implantcomprising a porous, non-porous, or gelatinous material; by topicalocular drops or ointments; or by a slow release device in the cul-de-sacor implanted adjacent to the sclera (transscleral) or within the eye.Intracameral injection may be through the cornea into the anteriorchamber to allow the agent to reach the trabecular meshwork.Intracanalicular injection may be into the venous collector channelsdraining Schlemm's canal or into Schlemm's canal. Further modes ofadministration include tablets, pills, and capsules.

Administration may be directly to the ear via, for example, topical oticdrops or ointments, slow release devices in the ear or implantedadjacent to the ear. Local administration includes otic intramuscular,intratympanic cavity and intracochlear injection routes ofadministration. Furthermore, agents can be administered to the inner earby placement of a gelfoam, or similar absorbent and adherent product,soaked with the interfering RNA against the window membrane of themiddle/inner ear or adjacent structure.

Administration may be directly to the lungs, via, for example, anaerosolized preparation, and by inhalation via an inhaler or anebulizer, for example

Subject: A subject in need of treatment for a Syk-related inflammatorycondition or at risk for developing a Syk-related inflammatory conditionis a human or other mammal having a Syk-related inflammatory conditionor at risk of developing a Syk-related inflammatory condition, such asallergic conjunctivitis, ocular inflammation, dermatitis, rhinitis,asthma, allergy, or mast-cell disease for example, associated withundesired or inappropriate expression or activity of Syk as citedherein.

Ocular structures associated with such disorders may include the eye,retina, choroid, lens, cornea, trabecular meshwork, iris, optic nerve,optic nerve head, sclera, aqueous chamber, vitreous chamber, ciliarybody, or posterior segment, for example.

Otic structures associated with such disorders may include the innerear, middle ear, outer ear, tympanic cavity or membrane, cochlea, orEustachian tube, for example.

Pulmonary structures associated with such disorders may include thenose, mouth, pharynx, larynx, bronchial tubes, trachea, carina (theridge separating the opening of the right and left main bronchi), andlungs, particularly the lower lungs, such as bronchioli and alveoli.

A subject may also be an otic cell, a lung cell, an ocular cell, cellculture, organ or an ex vivo organ or tissue.

Formulations and Dosage: Pharmaceutical formulations compriseinterfering RNAs, or salts thereof, of the invention up to 99% by weightmixed with a physiologically acceptable carrier medium such as water,buffer, saline, glycine, hyaluronic acid, mannitol, and the like.

Interfering RNAs of the present invention are administered as solids,solutions, suspensions, or emulsions. The following are examples ofpossible formulations embodied by this invention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Hydroxypropylmethylcellulose 0.5 Sodium chloride 0.8 BenzalkoniumChloride 0.01 EDTA 0.01 NaOH/HCl Qs pH 7.4 Purified water (RNase-free)Qs 100 Ml Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0 PhosphateBuffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 80 0.5Purified water (RNase-free) q.s. to 100% Interfering RNA up to 99;0.1-99; 0.1-50; 0.5-10.0 Monobasic sodium phosphate 0.05 Dibasic sodiumphosphate 0.15 (anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05Cremophor EL 0.1 Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4Purified water (RNase-free) q.s. to 100% Interfering RNA up to 99;0.1-99; 0.1-50; 0.5-10.0 Phosphate Buffered Saline 1.0Hydroxypropyl-β-cyclodextrin 4.0 Purified water (RNase-free) q.s. to100%

Generally, an effective amount of the interfering RNAs of embodiments ofthe invention results in an extracellular concentration at the surfaceof the target cell of from 100 μM to 100 nM, or from 1 nM to 50 nM, orfrom 5 nM to about 10 nM, or about 25 nM. The dose required to achievethis local concentration will vary depending on a number of factorsincluding the delivery method, the site of delivery, the number of celllayers between the delivery site and the target cell or tissue, whetherdelivery is local or systemic, etc. The concentration at the deliverysite may be considerably higher than it is at the surface of the targetcell or tissue. Topical compositions are delivered to the surface of thetarget organ one to four times per day, or on an extended deliveryschedule such as daily, weekly, bi-weekly, monthly, or longer, accordingto the routine discretion of a skilled clinician. The pH of theformulation is about pH 4-9, or pH 4.5 to pH 7.4.

Therapeutic treatment of patients with siRNAs directed against Syk mRNAis expected to be beneficial over small molecule treatments byincreasing the duration of action, thereby allowing less frequent dosingand greater patient compliance.

An effective amount of a formulation may depend on factors such as theage, race, and sex of the subject, the severity of the Syk-relatedinflammatory condition, the rate of target gene transcript/proteinturnover, the interfering RNA potency, and the interfering RNAstability, for example. In one embodiment, the interfering RNA isdelivered topically to a target organ and reaches spleen tyrosine kinasecontaining tissue at a therapeutic dose thereby ameliorating aSyk-related process.

Acceptable carriers: An acceptable carrier refers to those carriers thatcause at most, little to no ocular irritation, provide suitablepreservation if needed, and deliver one or more interfering RNAs of thepresent invention in a homogenous dosage. An acceptable carrier foradministration of interfering RNA of embodiments of the presentinvention include the cationic lipid-based transfection reagentsTransIT®-TKO (Minis Corporation, Madison, Wis.), LIPOFECTIN®,Lipofectamine, OLIGOFECTAMINE™ (Invitrogen, Carlsbad, Calif.), orDHARMAFECT™ (Dharmacon, Lafayette, Colo.); polycations such aspolyethyleneimine; cationic peptides such as Tat, polyarginine, orPenetratin (Antp peptide); or liposomes. Liposomes are formed fromstandard vesicle-forming lipids and a sterol, such as cholesterol, andmay include a targeting molecule such as a monoclonal antibody havingbinding affinity for endothelial cell surface antigens, for example.Further, the liposomes may be PEGylated liposomes.

The interfering RNAs may be delivered in solution, in suspension, or inbioerodible or non-bioerodible delivery devices. The interfering RNAscan be delivered alone or as components of defined, covalent conjugates.The interfering RNAs can also be complexed with cationic lipids,cationic peptides, or cationic polymers; complexed with proteins, fusionproteins, or protein domains with nucleic acid binding properties (e.g.,protamine); or encapsulated in nanoparticles. Tissue- or cell-specificdelivery can be accomplished by the inclusion of an appropriatetargeting moiety such as an antibody or antibody fragment.

For ophthalmic, otic, or pulmonary delivery, an interfering RNA may becombined with ophthalmologically, optically, or pulmonary acceptablepreservatives, co-solvents, surfactants, viscosity enhancers,penetration enhancers, buffers, sodium chloride, or water to form anaqueous, sterile suspension or solution. Solution formulations may beprepared by dissolving the interfering RNA in a physiologicallyacceptable isotonic aqueous buffer. Further, the solutions may includean acceptable surfactant to assist in dissolving the inhibitor.Viscosity building agents, such as hydroxymethyl cellulose, hydroxyethylcellulose, methylcellulose, polyvinylpyrrolidone, or the like may beadded to the compositions of the present invention to improve theretention of the compound.

In order to prepare a sterile ointment formulation, the interfering RNAis combined with a preservative in an appropriate vehicle, such asmineral oil, liquid lanolin, or white petrolatum. Sterile gelformulations may be prepared by suspending the interfering RNA in ahydrophilic base prepared from the combination of, for example,CARBOPOL°-940 (BF Goodrich, Charlotte, N.C.), or the like, according tomethods known in the art. VISCOAT® (Alcon Laboratories, Inc., FortWorth, Tex.) may be used for intraocular injection, for example. Othercompositions of the present invention may contain penetration enhancingagents such as cremephor and TWEEN® 80 (polyoxyethylene sorbitanmonolaureate, Sigma Aldrich, St. Louis, Mo.), in the event theinterfering RNA is less penetrating in the organ or tissue of interest.

Kits: Embodiments of the present invention provide a kit that includesreagents for attenuating the expression of an mRNA as cited herein in acell. The kit contains an siRNA or an shRNA expression vector. ForsiRNAs and non-viral shRNA expression vectors the kit also may contain atransfection reagent or other suitable delivery vehicle. For viral shRNAexpression vectors, the kit may contain the viral vector and/or thenecessary components for viral vector production (e.g., a packaging cellline as well as a vector comprising the viral vector template andadditional helper vectors for packaging). The kit may also containpositive and negative control siRNAs or shRNA expression vectors (e.g.,a non-targeting control siRNA or an siRNA that targets an unrelatedmRNA). The kit also may contain reagents for assessing knockdown of theintended target gene (e.g., primers and probes for quantitative PCR todetect the target mRNA and/or antibodies against the correspondingprotein for western blots). Alternatively, the kit may comprise an siRNAsequence or an shRNA sequence and the instructions and materialsnecessary to generate the siRNA by in vitro transcription or toconstruct an shRNA expression vector.

A pharmaceutical combination in kit form is further provided thatincludes, in packaged combination, a carrier means adapted to receive acontainer means in close confinement therewith and a first containermeans including an interfering RNA composition and an acceptablecarrier. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc., as will be readily apparent tothose skilled in the art. Printed instructions, either as inserts or aslabels, indicating quantities of the components to be administered,guidelines for administration, and/or guidelines for mixing thecomponents, can also be included in the kit.

The ability of Syk interfering RNA to knock-down the levels ofendogenous Syk expression in, for example, human corneal epithelialcells is evaluated in vitro as follows. Transformed human cornealepithelial cells, for example, the CEPI-17 cell line (Offord et al.(1999) Invest Ophthalmol Vis Sci. 40:1091-1101), are plated 24 h priorto transfection in KGM keratinocyte medium (Cambrex, East Rutherford,N.J.). Transfection is performed using DharmaFECT™ 1 (Dharmacon,Lafayette, Colo.) according to the manufacturer's instructions at Sykinterfering RNA concentrations ranging from 0.1 nM-100 nM. Non-targetingcontrol interfering RNA and lamin A/C interfering RNA (Dharmacon) areused as controls. Target mRNA levels are assessed by qPCR 24 hpost-transfection using, for example, TAQMAN® forward and reverseprimers and a probe set that encompasses the target site (AppliedBiosystems, Foster City, Calif.). Target protein levels may be assessedapproximately 72 h post-transfection (actual time dependent on proteinturnover rate) by western blot, for example. Standard techniques for RNAand/or protein isolation from cultured cells are well-known to thoseskilled in the art. To reduce the chance of non-specific, off-targeteffects, the lowest possible concentration of Syk interfering RNA isused that produces the desired level of knock-down in target geneexpression.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

Interfering RNA for Specifically Silencing SYK in 293FT Cells

The present study examines the ability of SYK-interfering RNA to knockdown the levels of endogenous SYK protein expression in cultured 293FTcells.

Transfection of 293FT cells (Invitrogen, Carlsbad, Calif.) wasaccomplished using standard in vitro concentrations (0.1-10 nM) of SYKsiRNAs, siCONTROL RISC-free siRNA #1, or siCONTROL Non-targeting siRNA#2 (NTC2) and DHARMAFECT® #1 transfection reagent (Dharmacon, Lafayette,Colo.). All siRNAs were dissolved in 1×siRNA buffer, an aqueous solutionof 20 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mM MgCl₂. Control samplesincluded a buffer control in which the volume of siRNA was replaced withan equal volume of 1×siRNA buffer (-siRNA). Western blots using ananti-SYK antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) wereperformed to assess SYK protein expression. The SYK siRNAs aredouble-stranded interfering RNAs having specificity for the followingtargets: siSYK #2 targets the sequence AAUGCCUUGGUUCCAUGGA (SEQ ID NO:44); siSYK #5 targets the sequence GGAAUAAUCUCAAGAAUCA (SEQ ID NO: 45);siSYK #6 targets the sequence GAACUGGGCUCUGGUAAUU (SEQ ID NO: 46); siSYK#8 targets the sequence GAACAGACAUGUCAAGGAU (SEQ ID NO: 47). As shown bythe data of FIG. 1, siSYK #5, siSYK #6, and siSYK #8 siRNAs reduced SYKprotein expression significantly at the 10 nM and 1 nM concentrationsrelative to the control siRNAs, but exhibited reduced efficacy at 0.1nM. The siSYK #6, and siSYK #8 siRNAs were particularly effective. ThesiSYK #2 siRNA was ineffective at all three concentrations.

What is claimed is:
 1. A method of attenuating expression of Syk mRNA ofa subject, comprising: administering to the subject a compositioncomprising an effective amount of interfering RNA (iRNA) having a lengthof 19 to 49 nucleotides and a pharmaceutically acceptable carrier, theinterfering RNA comprising a region selected from the group consistingof: a region of at least 13 contiguous nucleotides having at least 90%sequence complementarity to, or at least 90% sequence identity with, thepenultimate 13 nucleotides of the 3′ end of an mRNA corresponding to anyone of SEQ ID NO: 21-22, 25, 29, 35-36, 39, and 42; a region of at least14 contiguous nucleotides having at least 85% sequence complementarityto, or at least 85% sequence identity with, the penultimate 14nucleotides of the 3′ end of an mRNA corresponding to any one of SEQ IDNO: 21-22, 25, 29, 35-36, 39, and 42; and, a region of at least 15, 16,17, or 18 contiguous nucleotides having at least 80% sequencecomplementarity to, or at least 80% sequence identity with, thepenultimate 15, 16, 17, or 18 nucleotides, respectively, of the 3′ endof an mRNA corresponding to any one of SEQ ID NO: 21-22, 25, 29, 35-36,39, and 42, wherein the expression of Syk mRNA is attenuated thereby. 2.The method of claim 1, wherein the interfering RNA is an shRNA.
 3. Themethod of claim 1, wherein the composition is administered via anaerosol, buccal, dermal, intradermal, inhaling, intramuscular,intranasal, intraocular, intrapulmonary, intravenous, intraperitoneal,nasal, ocular, oral, otic, parenteral, patch, subcutaneous, sublingual,topical, or transdermal route.
 4. The method of claim 1, wherein theinterfering RNA is administered via in vivo expression from anexpression vector capable of expressing the interfering RNA.
 5. Themethod of claim 1, wherein the interfering RNA is at least one of anmiRNA or an siRNA.
 6. The method of claim 1, wherein the subject is ahuman and the human has a Syk-related inflammatory condition or is atrisk of developing a Syk-related inflammatory condition.
 7. A method oftreating a Syk-related inflammatory condition in a subject in needthereof, comprising: administering to the subject a compositioncomprising an effective amount of interfering RNA having a length of 19to 49 nucleotides, and a pharmaceutically acceptable carrier, theinterfering RNA comprising a sense nucleotide strand, an antisensenucleotide strand, and a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides; wherein the antisense strandhybridizes under physiological conditions to a portion of mRNA selectedfrom the group consisting of mRNA corresponding to SEQ ID NO:1comprising nucleotide 1471, 1472, 1680, 1880, 2473, 2509, or 2558, and,mRNA corresponding to SEQ ID NO:1 comprising nucleotide 1698, whereinthe Syk-related inflammatory condition is treated thereby.
 8. The methodof claim 7, wherein the subject is a human and the human has aSyk-related inflammatory condition or is at risk of developing aSyk-related inflammatory condition.
 9. The method of claim 7, whereinthe sense nucleotide strand and the antisense nucleotide strand areconnected by a loop nucleotide strand.
 10. The method of claim 7,wherein the composition is administered via an aerosol, buccal, dermal,intradermal, inhaling, intramuscular, intranasal, intraocular,intrapulmonary, intravenous, intraperitoneal, nasal, ocular, oral, otic,parenteral, patch, subcutaneous, sublingual, topical, or transdermalroute.
 11. The method of claim 7, wherein the interfering RNA isadministered via in vivo expression from an expression vector capable ofexpressing the interfering RNA.
 12. A method of attenuating expressionof Syk mRNA of a subject, comprising: administering to the subject acomposition comprising an effective amount of a single-strandedinterfering RNA having a length of 19 to 49 nucleotides, and apharmaceutically acceptable carrier, wherein the single-strandedinterfering RNA hybridizes under physiological conditions to a portionof mRNA selected from the group consisting of mRNA corresponding to SEQID NO:1 comprising nucleotide 1471, 1472, 1680, 1880, 2473, 2509, or2558, and, mRNA corresponding to SEQ ID NO:1 comprising nucleotide 1698,and the interfering RNA has a region of at least near-perfect contiguouscomplementarity with the hybridizing portion of mRNA corresponding toSEQ ID NO:1, wherein the expression of Syk mRNA is thereby attenuated.13. The method of claim 12, wherein the composition is administered viaan aerosol, buccal, dermal, intradermal, inhaling, intramuscular,intranasal, intraocular, intrapulmonary, intravenous, intraperitoneal,nasal, ocular, oral, otic, parenteral, patch, subcutaneous, sublingual,topical, or transdermal route.
 14. The method of claim 12, wherein theinterfering RNA is administered via in vivo expression from anexpression vector capable of expressing the interfering RNA.
 15. Themethod of claim 12, wherein the interfering RNA is at least one of anmiRNA or an siRNA.
 16. A method of treating ocular inflammation orconjunctivitis in a subject in need thereof, comprising: administeringto the subject a composition comprising a double stranded siRNA moleculethat comprises a nucleotide sequence corresponding to any one of SEQ IDNO: 21-22, 25, 29, 35-36, 39, and 42 and down regulates expression of aSyk gene via RNA interference, wherein: each strand of the siRNAmolecule is independently about 19 to about 27 nucleotides in length;and one strand of the siRNA molecule comprises a nucleotide sequencehaving substantial complementarity to an mRNA corresponding to the Sykgene so that the siRNA molecule directs cleavage of the mRNA via RNAinterference.
 17. The method of claim 16, wherein the composition isadministered via an aerosol, buccal, dermal, intradermal, inhaling,intramuscular, intranasal, intraocular, intrapulmonary, intravenous,intraperitoneal, nasal, ocular, oral, otic, parenteral, patch,subcutaneous, sublingual, topical, or transdermal route.
 18. The methodof claim 16, wherein the interfering RNA is administered via in vivoexpression from an expression vector capable of expressing theinterfering RNA.
 19. The method of claim 16, wherein each strand of thesiRNA molecule is independently about 19 nucleotides to about 25nucleotides in length or independently about 19 nucleotides to about 21nucleotides in length.
 20. A composition comprising an interfering RNAhaving a length of 19 to 49 nucleotides, and comprising a nucleotidesequence corresponding to any one of SEQ ID NO: 21-22, 25, 29, 35-36,39, and 42, or a complement thereof; and a pharmaceutically acceptablecarrier.
 21. The composition of claim 20, wherein the interfering RNA isat least one of an shRNA, an siRNA, or an miRNA.